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Fig. 4. 14-3-3ß but not 14-3-3 ${sigma}$ efficiently redistributes CDC25B from the nucleus to the cytoplasm. HEK293 cells were transfected with FLAG-tagged CDC25B in combination with empty vector, myc-tagged 14-3-3ß or myc-tagged 14-3-3 ${sigma}$ , followed by immunostaining with anti-FLAG antibodies to detect the subcellular localization of CDC25B and with anti-myc antibodies to detect cotransfected 14-3-3 proteins. Analyses showed that more than 95% of the cells that expressed CDC25B also expressed the co-transfected 14-3-3 proteins. (A) Exemplary images, showing how the subcellular distribution of CDC25B was evaluated: N>C, predominantly nuclear; N=C diffuse; N<C, predominantly cytoplasmic. (B) Wild-type CDC25B was co-transfected with empty vector (WT), myc-tagged 14-3-3ß (WT+14-3-3ß) or myc-tagged 14-3-3 ${sigma}$ (WT+14-3-3 ${sigma}$ ) to quantify the subcellular distribution of CDC25B. Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments. (C) Transfection with wild-type CDC25B alone (WT), S309A mutant of CDC25B alone (S309A) and mutant S309A in combination with myc-tagged 14-3-3ß (S309A+14-3-3ß) or myc-tagged 14-3-3 ${sigma}$ (S309A+14-3-3 ${sigma}$ ). Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments. (D) Transfection with wild-type CDC25B alone (WT), S216A mutant of CDC25B alone (S216A) and mutant S216A in combination with myc-tagged 14-3-3ß (S216A+14-3-3ß) or myc-tagged 14-3-3 ${sigma}$ (S216A+14-3-3 ${sigma}$ ). Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments.

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